PCR: Aplikasi dan Design

Eksperimen
Iman Permana M
Kimia FMIPA UNPAD
 PCR
(Polymerase Chain reaction)

Mesin PCR
 Peralatan umum

Mikropipet dan mikrotip

Microcentrifuge/
Refrigerred microcentrifuge
 Peralatan Umum

• Laminar air flow

• Blower
 PCR
 Polymerase Chain Reaction
 Allows amplification from nucleic acid
templates
 enzymatic reaction
– relies on fidelity of enzyme to produce a
complementary sequence to the template
strand in the presence of primers and
deoxyribonucleotides
 Reaction
Once a round is completed, the
new DNA products are
subjected to denaturation and
Double stranded re-enter the amplification
template is Denature procedure.
dissociated by Re-enter cycle
heating to 95oC

Anneal Primers
On the final cycle, finish
with an extension step.

Extend

Temperature is decreased to
Extension from primer sites at optimal temperature of 68-74oC.
optimal hybridisation
Extension time varies according to product size: enzyme activity
temperature for duplex
averages 1Kb per min. 68 oC is preferred for longer templates to
formation in the presence of
conserve enzyme activity over multiple cycles.
extension primers.
 Reaction
Satu putaran sudah lengkap,
dua pasang DNA diperoleh
sebagai produk , yang
selanjutnya didenaturasi dan
masuk ke pengulangan
Pembukaan dua
Denaturasi prosedur amplifikasi.
untai templat
DNA oleh Pengulangan siklus
pemanasan
sampai 95oC.

Penempelan primer (Annealing)

Penyelesain dalam satu
siklus.

Perpanjangan

Temperatur diturunkan Perpanjangan primer pada temperatur optimal 68-

sampai temperatur 74oC. Variasi waktu perpanjangan berdasarkan
hibridisasi optimal untuk perbandingan ukuran produk : aktivitas enzim rata-rata
pembentukan duplek 1Kb per menit. 68 oC.
primer dengan DNA
templat secara antiparalel .
 Reaction Profile
Components of the reaction are usually in excess (primers, dNTPs and
enzyme). Final yield depends on the template composition and concentration.
GC rich templates can be more difficult to PCR efficiently owing to formation
of strong secondary structures at the annealing temperature and require
modified protocols to maintain an open DNA structure: for example addition
of DMSO.

log[DNA]
In the early phase of the PCR cycling, the yield is limited by the
concentration of the template in the reaction, and the increase in
product per round will be less than 2-fold.The reaction then
reaches a linear phase where the amplification is approximately
2-fold. Finally, exhaustion of components/ decreasing enzyme
activity result in declining efficiency of amplification and a
plateau. Most PCR reactions will be stopped during the late linear
phase.

Cycle number
 PCR
Komponen PCR

• DNA templat
• Sepasang oligonukleotida primer
• Campuran dNTP (dATP, dGTP, dTTP dan dCTP)
• Enzim DNA polimerase termostabil
• Buffer
• Program

DNA templat
primer
 KOMPONEN PCR
Templat DNA  keutuhan &  DNA
Primer :
 Desain primer optimal :
- Panjang primer  20 nukleotida
- % GC = 50 – 60 %
- Tm = 60 – 70 oC
- Minimalkan self complementary
terutama pada ujung 3’ primer
 Kons. & Komposisi primer setimbang (@ 0,1-0,4M)
dNTP :
 Kons. & Komposisi dNTP optimal (@ 0,1-0,4mM)
......komp. PCR

Enzim DNA polimerase:

 3’  5’ eksonuklease (proofreading)
 non proofreading (mis: Taq polimerase)
 jumlah enzim optimal
Ion Mg2+  Sensitifitas & spesifitas
Bufer PCR dg pH optimal (8,8-9,2) Fidelitas & Tdntrsi
 Template
 Template quality DNA from crude preparation protocols may be sheared. Such DNA cannot be
used for methods such as Southern blotting, which require high quality and high
molecular weight substrates. However, provided the PCR product is short, partial

– fragmentation
fragmentation is not an issue. Sufficient numbers of molecules remaining intact
between the primer binding sites will be present.
This ability to retrieve information from degraded starting material makes PCR

– source
ideal in cases of forensic analysis, or molecular analysis of genes with putative
mutations from old pathological specimens.

Template concentration
PCR is sensitive enough to amplify from small amounts of starting

 material. Normally, a 50l PCR will contain 50ng of genomic DNA, or

~1.5 x 104 copies of the target and should give a signal on an agarose
gel after 30 cycles. However, modified protocols using a random primed

– copy number
pre-amplification of template can be used in conjunction with a second
targeted PCR to allow detection of sequences from single cells,
including haploid germ cells

 Template sequence characteristics

– G+C content
High GC content, or internal complementarity of sequence can

– structural constraints
reduce PCR efficiency if the template can form stable secondary or
tertiary structures at the temperatures used for primer annealing
and extension, as the enzyme cannot proceed along the template.
This can be overcome by the addition of reagents that inhibit the
folding of single-stranded DNA.
 Enzymes
 Original PCR carried out with DNA polI
Klenow fragment
– Problematic

The Klenow fragment of DNA polymerase I from E. coli is not thermostable. Early
PCR reactions required an optimum extension temperature of 37oC, and additional
enzyme was added to the reaction after every round of denaturation. Like all
enzymes, once the glycerol content of the reaction mix exceeds 10%, enzyme
efficiency is dramatically reduced. In addition, the reaction volume was altered after
each round of PCR by the addition step. Such PCR protocols were difficult to
perform (heating blocks/ water baths at the 3 temperatures required), had high risk of
contamination (opening tube every round), and were labour intensive (manual
intervention at every step)
 Enzymes
 Original PCR carried out with DNA polI Klenow
fragment
– Problematic
 New polymerases from bacteria tolerant of high
temperatures eg Thermus aquaticus (Taq)
 Fidelity
 Enzyme concentration
The introduction of enzymes from bacteria tolerant
of high temperatures made PCR a more viable
procedure:
1. Single set up with no intervention, so less

– usually 0.01-0.02 units/l contamination

2. Development of machines incorporating dry blocks
or plunge baths and programs for contolling the
applied temperature at each step in the cycling
process.
3. Combinations of enzymes to improve fidelity
through proof-reading.
 Primers
 Primer design must consider Internal secondary structure in

– melting temperature of duplex primers, or dimer formation

between primer pairs reduces the
free concentration of primers in
 primer composition and primer length the reaction mixture and can
compete with template-primer

– primer compatibility
duplexes for enzyme.

 temps, internal homology, dimer formation

– primer sequence homologies
 BLAST analysis Primers must not lie within common repeat elements, or regions with high
sequence identity e.g. gene families /motifs, as competition for binding sites on a
complex template will reduce the yield of the correct product.

 Most primer design using specialist programs

 Use at 1M final concentration
 Design Primer
 Panjang primer 15-30 nukleotida (primer yang lebih
panjang memiliki spesifisitas yang lebih tinggi)
 % GC = 40 – 60 %
 Nukleotida C dan G harus terdistribusi disepanjang
primer
 Lebih dari 3 nukleotida G atau C diujung 3’ menghasilkan
primer yang non spesifik
 Minimalkan self complementary terutama pada ujung 3’
primer serta dimerisasi antar primer
 Selisih Tm antar primer tidak lebih dari 5oC
 Jika primer <25 nukleotida, Tm = 4 (G+C) + 2 (A+T)
 Jika Tm >25 nukleotida dengan Pendekatan beberapa
software: DNAstar, Perlprimer, oligoexplorer dan
FastPCR.
 Buffers
 Salts: K/ Mg
– Normally only Mg is titrated- essential for optimal
enzyme activity and product yield
 free Mg for enzyme activity
 excess Mg causes in non-specific amplification
 Detergents
– essential for enzyme activity
 Modifiers
– eg DMSO to reduce hydrogen bonding
– dNTP analogues digested
dUTP can be introduced in place of dTTP. Product with dUTP can be
using UNG (Uracil-N glycosylase). This is useful where routine
PCRs are carried out on a large scale, or in end-point applications such
as allele detection. The pretreatment of reactions with UNG reduces
cross-contamination of the template from previous products.
 Product
 Product size
– most standard enzymes will produce
fragments up to 3 Kb in size
Specialist kits with thermostable proof reading enzymes and

 Sequence composition
reagents designed to give longer products are now available.
These can amplify fragments up to 50Kb in length from
complex templates.

– G+C content may hinder extension from the

primer binding sites
 Programs
 Initial denaturation 95oC (3-5min)
 Denaturation (15-30s)
– template
 Anneal (20-30s)
– Duplex melting temp
 Extend (from 30s)
– Length of template (15 bases/s)
– Temp
 Final extension and hold A final extension at 72oC will ensure that all
the molecules in the final product mixture are
full length. The hold is normally at 4oC.
Products are then stored in a refrigerator or
freezer before analysis.
 Prosedur PCR :
Kondisi PCR :
• Denaturasi awal : 94oC, 1 menit
• Denaturasi : 94oC, 1 menit
• Annealing : 50oC, 1 menit
• Polimerasi : 72oC, 1 menit
• Polimerasi akhir : 72oC, 4 menit
• Siklus : 30
• Enzim : 1,25 unit Taq Polimerase
• dNTP : 0,8 mM dNTP total
• MgCl2 : 1,5 mM
• Primer : @. 0,4 M
 Types of Template
 Genomic DNA
Genomic DNA may be extracted from individuals (e.g. patient

 Cloned DNA
samples, bacteria, viruses, plants etc.), cell lines, somatic cell
hybrids, radiation hybrids.

 RNA/ cDNA Cloned DNA can be extracted from large insert genomic
clones such as YACs, BACs, PACs, cosmid, phage, or, small
insert clones such as plasmids.

RNA templates are usually converted to cDNA templates prior

to PCR. Such templates can be used in real-time PCR
experiments to quantitate relative expression of mRNA
compared with control transcripts, or in reverse-transcription
studies coupled to gene specific PCR for investigation of
mRNA structure and coding potential.
 Siklus PCR :
 Denaturasi DNA template: 94-96oC
 Annealing  Tannealing = (Tm – 5)oC
 Extension suhu ~ aktivitas optimal
enzim DNA polimerase
Waktu ~panjang fragmen
 Siklus PCR

Cycle 1 Cycle 2 Cycle 3

94 oC 94 oC 94 oC
T
72 oC 72 oC 72 oC

55 oC 55 oC 55 oC

Time (seconds)
Perbanyakan DNA : secara
eksponensial

16

220
 -
P
C Polimerisasi DNA
Template
BASA’ BASA’
BASA BASA
dNTP
3’
Primer 3’  C5’ OH
5’ C P 
P -

M2+ C M2+ 
P
A B -
D882
-
C
DNA polimerase C
D705
 Mekanisme PCR ( animasi )
Mg2+
5‘ 3‘ 3‘
5‘

5‘
DNA
3‘ 3‘ 5‘

94oC (Denaturasi)
5‘ 3‘

3‘ 5‘
5‘ 3‘
Mg2+

Mg2+ Mg2+

5‘ 3‘

3‘ 5‘

3‘ 5‘
3‘
5‘

65oC (Annealing) 3‘ 5‘

Mg2+ Mg2+

3‘
5‘
5‘ 3‘

3‘ 5‘
3‘ 5‘
Primer 5‘
3‘
Primer
5‘
Prod. 3‘ 5‘

5‘ 3‘
3‘ = (2n-2n)x 5‘

3‘ 5‘
3‘
5‘

72oC (extension) 3‘ 5‘
 Optimasi Suhu Annealing
5‘ 3‘

3‘ 5‘
Primer Primer 3‘ 5‘
5‘
Primer
Primer Tidak ada Produk
5‘

3‘ 5‘

Annealing >> Tm
3
5‘

5‘ 3‘ 3‘ 5

3‘
Annealing 3‘ 5‘ 5‘
Primer
Tm-5OC Primer 3‘ 5‘
5‘
3‘
5‘
3‘ 5‘
3‘ 5‘
3‘
5‘

Annealing << Tm 3‘ 5‘

5‘ 3‘

3‘ 5‘
5‘ 3‘
3‘
5‘
3‘ 5‘ 3‘ 5‘
3‘ 5‘ Produk
Primer Primer 5‘ 3‘ non spesifik
Primer Primer 3‘ 5‘
5‘ 5‘ 3‘
5‘

3‘ 5‘ 5‘
3‘
 Peranan Ion Mg2+
Meningkatkan kinerja enzim Taq pol
Meningkatkan Tm primer & template DNA
Meningkatkan kelarutan dNTP
Meningkatkan sensitifitas / yield
 Peranan ion Mg 2+ - animasi -
2+ 2+ 2+
dNTP kelarutan besar 2+ 2+ 2+ 2+
2+
2+ 2+ 2+
2+
2+ 2+ 2+
2+

2+ 2+ 2+ 2+
2+ 2+
Mg2+
(mengikat ion Mg2+)
2+ 2+
2+ 2+ 2+
Mg2+2+ 2+
5 3 2+ 2+

2+ 2+ ‘ 5 3
‘ Mg2+
2+
3 5 2+ 2+
‘ ‘
2+ ‘2+ 2+
‘
2+
32+ 2+
2+ 2+ 5
2+
2+
2+ 2+
2+ 2+ ‘ 2+ 2+
+ ‘
2+ 2+ 2+
2+ 2+ 2+ 2+ 2
2+
2+ 2+ 2+
2+ 2+ 2+
2+ 2+
2+ 2+
2+
3
2+
‘
5 3 2+ 2+
2+ 2+2+
2+ 2+ 2+
2+ ‘ 2+ Mg2+‘ 2+2+ 2+
5
2+ 2+
2+ 3
2+ 5 2+
2+ 2+
2+ 2+ 2+ ‘ 2+
2+
2+ 2+
2+ 2+
2+ 2+
‘ 2+
2+
2+ ‘ 2+ 2+
3
2+
2+
2+
2+ 5
2+2+ 2+ 2+ 2+
2+ 2+
2+
2+ 2+ 2+ ‘ Mg2+ 2+ 2+ Mg2+‘
2+ 2+2+ 2+ 2+
2+ 2+ 2+ 2+
2+ 2+ 2+ 2+2+ 2+
2+ 2+ 2+ 2+
2+ 2+
2+ 2+ 2+ 2+
2+ 2+ 2+ 2+ 2+ 2+ 2+
2+ 2+ 2+
2+ 2+ 2+ 2+ 2+ 2+
2+ 2+ 2+ 2+
2+ 2+ 2+ 2+ 2+
2+ 2+ 2+
2+ 2+ 2+
2+
2+ 2+
2+ 2+
2+ 2+ 2+
2+
2+

dNTP kelarutan kecil (tanpa ion Mg2+)

 Application of PCR
 Sequence Tagged sites (STS)
– Genomic, RH, and cloned DNA
– For order information, library screening
– SNP discovery
STS have proved extremely useful in the development of overlapping
genomic clones (contigs) from organism specific libraries prior to
sequencing of the genome. Initially random STS were generated, but
directed STS from clone ends have also hastened this assembly

 Genomic DNA procedure. In less developed phases of genome analysis, radiation

hybrids (Rh panels) are used to order genes within a chromosomal
framework relative to known mapped markers.

 cDNA
STS designed from sequence information can also be used to re-
sequence the equivalent region of the genome from different
individuals within a species to look at single nucleotide polymorphism
(SNP) frequency, or between species to define evolutionary
differences.
 Genomic DNA

Cloning
e.g.Functional promoter assays

Mutation
studies
e.g patient material

Comparative SNPs
studies Discovery via
e.g. genome evolution, sequencing
classification of bacterial
strains

Sequencing
Quantitative PCR projects
looking for copy number variation:
often PCR based sequencing
Allele specific PCR
OK for 1:2 ratio, not for 2:3 etc SNP typing of individuals or populations
methods
 RNA/cDNA

In situ
PCR
using fixed tissue samples
RT-PCR to determine where/ which
reverse transcription cell types in an organ
coupled to end point produce transcripts.
PCR or real-time Require primers
PCR protocols for complementary to the +
transcription analysis Differential strand template

RACE- rapid Display

Comparison of expression profiles
amplification between 2 states, e.g. cell line
stimulated with a hormone v untreated
of cDNA ends
useful for extension of ORF from
a small amount of known
sequence information, or
completion of 5’ and 3’ ends of a
transcript.
 Cloned DNA

Small inserts Large inserts

e.g. plasmids e.g BAC/ PAC/ YAC

Gene
structure
e.g. sizing introns and
determing intron-exon
boundaries based on
knowledge of mRNA

Whole insert STS content

End clone Mapping and order information
amplification
can be used for insert analysis, or recovery
to produce material for spotting on development of new STS, and
microarrays contig confirmation
 Experimental controls
 Positive control (sample with known
outcome)
 Negative control (e.g. water only, to
assess contamination)
 Genomic control for cDNA based PCR
studies (usually primers flank an intron in
genomic PCR, so product sizes differ from
the cDNA template)
 Specialist PCR
 Long range PCR For amplification of fragments 3-50Kb: specialist enzyme, buffer and cycling conditions

High fidelity PCR

Especially for products that will be subcloned for e.g. expression studies
 where mutations could be deleterious

Allele specific PCR

For genotyping individuals or populations: can use modified standard PCR
 techniques, or reporter based assays

 Quantitation For gene copy number or for relative gene expression in mRNA samples

 ISH (in situ hybridisation) and PRINS (primed

in situ)
 Cross species
 Whole genome amplification
 Sequencing Modified PCR with ddNTPs to
terminate the chain extension reactions.
 Troubleshooting guide
 No PCR product observed
 Reaction mixture incomplete (always perform a positive
control)
 Insufficient cycles
 Annealing temperature too high
 Not enough template
 Suboptimal denaturation temperature and length
 Suboptimal extension time (especially for long-distance PCR)
 Suboptimal reaction components (Mg2+, dNTP, primers
concentrations)
 Difficult PCR (Some PCR reactions may require the addition of
PCR enhancers)
 Poor amplimer design (Check design of primers, e.g.
sequence, GC content, length, Tm, lack of complementary)
 Multiple products
 Too many cycles
 Annealing temperature too low

 PCR products smeared

 Too many cycles
 Denaturation temperature too low
 Extension time too long
 Template degraded
 Too much enzyme, Mg2+ or template
 Enhancers of PCR
 Formamide (5%)
 Dimethyl sulfoxide (DMSO, <10%)
 Tetramethylammoniumchloride (TMAC, 10-100μm)
 Polyethylene glycol 6000 (PEG, 5-15%)
 Glycerol (10-15%)
 E. coli single-strand DNA binding protein (SSB) (5
μg/mL)
 Nucleotide analogs

 7 deaza-dGTP (reduces DNA secondary structure where

G-rich regions affect PCR efficiency and the resolution of
sequencing reactions
 Fluorescent-dye-labeled ddNTPs (used in automated
DNA sequencers)
 [α32P]dNTPs (radiolabeling of PCR products)
 [α35S]dNTPs (radiolabeling of PCR products)
 Biotin-11/16/21-dUTP (nonradioactive labeling of PCR
products)